Distribution by Scientific Domains
Distribution within Life Sciences

Terms modified by Phosphoinositide

  • phosphoinositide metabolism

  • Selected Abstracts

    In vitro evaluation of reactive astrocyte migration, a component of tissue remodeling in glaucomatous optic nerve head

    GLIA, Issue 3 2001
    Gülgün Tezel
    Abstract In order to improve understanding of remodeling events in the glaucomatous optic nerve head, the migration of optic nerve head astrocytes was studied in vitro. Since elevated intraocular pressure is an important stress factor identified in glaucomatous eyes, optic nerve head astrocytes were incubated under physical stress created by elevated hydrostatic pressure. In addition, they were incubated in the presence of a chemical stimulus, lipolysaccharide (LPS). Migration of reactivated astrocytes in the presence of these stressors was examined using chambers in which cell migration through extracellular matrix-coated pores is only possible following proteolytic digestion of the matrix. We observed that the migratory ability of optic nerve head astrocytes was approximately 4,6 times greater following exposure to elevated hydrostatic pressure or LPS for up to 48 h. Phosphoinositide 3-kinase, protein kinase C, and tyrosine kinase were found to be involved in the signal transduction for activated migration of optic nerve head astrocytes in response to elevated hydrostatic pressure or LPS. In addition, we observed that the stress-induced migration of optic nerve head astrocytes, which is accompanied by proteolytic degradation, resulted in the formation of culture cavities containing mucopolysaccharides. These in vitro findings provide a clearer understanding of the pathophysiologic mechanisms of characteristic tissue remodeling events that occur, in vivo, in the glaucomatous optic nerve head. GLIA 34:178,189, 2001. © 2001 Wiley-Liss, Inc. [source]

    Phosphoinositide 3-kinase signalling in lung disease: leucocytes and beyond

    IMMUNOLOGY, Issue 4 2007
    David A. Medina-Tato
    Summary The family of lipid kinases termed phosphoinositide-3-kinase (PI3K) is known to contribute at multiple levels to innate and adaptive immune responses, and is hence an attractive target for drug discovery in inflammatory and autoimmune disease, including respiratory diseases. The development of isoform-selective pharmacological inhibitors, targeted gene manipulation and short interfering RNA (siRNA) target validation have facilitated a better understanding of the role that each member of this family of kinases plays in the physiology and pathology of the respiratory system. In this review, we will evaluate the evidence for the roles of specific PI3K isoforms in the lung and airways, and discuss their potential as targets for novel drug therapies. [source]

    Phosphoinositide 3-kinase is not overexpressed in melanocytic lesions

    Rajendra S. Singh
    Background:, Although various studies have stressed the role of phosphatase and tensin homologue deleted on chromosome 10 (PTEN)-PI3K-AKT pathway in the progression of melanocytic lesions, little is known about the expression pattern of PI3K in these lesions. Objective:, To investigate the expression pattern of PI3K in benign and dysplastic nevi, primary melanomas, and metastatic melanomas and the role of PTEN and PI3K in melanocytic tumor progression. Methods:, Tissue microarrays were constructed using formalin-fixed, paraffin-embedded archival tissue blocks from 89 melanocytic lesions: 17 benign nevi, 18 dysplastic nevi, 23 primary melanomas, and 31 metastatic melanomas. Expression of PTEN and PI3K (p85 and p110 subunits) was evaluated immunohistochemically, and the number of cells and labeling intensity were assessed semiquantitatively. Results:, Both benign and dysplastic nevi showed strong cytoplasmic staining with PTEN, which was subsequently less in melanomas and completely lost in the metastatic lesions. Eleven of 17 (64%) benign nevi, seven of 10 (70%) dysplastic nevi, four of 23 (17%) primaries, and one of 31 (3%) visceral or lymph node metastasis showed strong positivity. Loss of PTEN expression from benign and dysplastic nevi to melanoma was statistically significant (p = 0.001). Although few cells showed reactivity for phosphoinositide 3-kinase (PI3 kinase)-p85 subunit, strong positivity was not detected in the cytoplasm of benign, malignant, or metastatic lesions, except for a single visceral metastasis. Three of 13 (23%) nevi showed positivity for the p110 subunit. No positivity was observed in the dysplastic nevi. Two of 22 (9%) melanomas, one of 14 (7%) visceral metastasis, and three of 12 (25%) lymph node metastasis showed strong positivity. There was no statistical difference in PI3 kinase expression in benign and malignant melanocytic lesions (p = 0.2). Conclusion:, PI3K is not overexpressed in melanocytic lesions. [source]

    Pathogen trafficking pathways and host phosphoinositide metabolism

    Stefan S. Weber
    Summary Phosphoinositide (PI) glycerolipids are key regulators of eukaryotic signal transduction, cytoskeleton architecture and membrane dynamics. The host cell PI metabolism is targeted by intracellular bacterial pathogens, which evolved intricate strategies to modulate uptake processes and vesicle trafficking pathways. Upon entering eukaryotic host cells, pathogenic bacteria replicate in distinct vacuoles or in the host cytoplasm. Vacuolar pathogens manipulate PI levels to mimic or modify membranes of subcellular compartments and thereby establish their replicative niche. Legionella pneumophila, Brucella abortus, Mycobacterium tuberculosis and Salmonella enterica translocate effector proteins into the host cell, some of which anchor to the vacuolar membrane via PIs or enzymatically turnover PIs. Cytoplasmic pathogens target PI metabolism at the plasma membrane, thus modulating their uptake and antiapoptotic signalling pathways. Employing this strategy, Shigella flexneri directly injects a PI-modifying effector protein, while Listeria monocytogenes exploits PI metabolism indirectly by binding to transmembrane receptors. Thus, regardless of the intracellular lifestyle of the pathogen, PI metabolism is critically involved in the interactions with host cells. [source]

    Several signaling pathways are involved in the control of cattle oocyte maturation

    Céline Vigneron
    Abstract The main limit of in vitro production of domestic mammal embryos comes from the low capacity of in vitro matured oocytes to develop after fertilization. As soon as they are separated from follicular environment, oocytes spontaneously resume meiosis without completion of their terminal differentiation. Roscovitine (ROS), an inhibitor of M-phase promoting factor (MPF) kinase activity reversibly blocks the meiotic resumption in vitro. However, in cattle maturing oocytes several cellular events such as protein synthesis and phosphorylation, chromatin condensation and nuclear envelope folding escape ROS inhibition suggesting the alternative pathways in oocyte maturation. We compared the level of synthesis and phosphorylation of several protein kinases during bovine cumulus oocyte complex (COC) maturation in vitro in the presence or not of epidermal growth factor (EGF) and ROS. We showed that during the EGF-stimulated maturation, ROS neither affected the decrease of EGF receptor (EGFR) nor did inhibit totally its phosphorylation in cumulus cells and also did not totally eliminate tyrosine phosphorylation in oocytes. However, ROS did inhibit the Phosphoinositide 3-kinase (PI3) activity when oocytes mature without EGF. Accumulation of Akt/PKB (protein kinase B), JNK1/2 (jun N-terminal kinases) and Aurora-A in oocytes during maturation was not affected by ROS. However, the phosphorylation of Akt but not JNKs was diminished in ROS-treated oocytes. Thus, PI3 kinase/Akt, JNK1/2 and Aurora-A are likely to be involved in the regulation of bovine oocyte maturation and some of these pathways seem to be independent to MPF activity and meiotic resumption. This complex regulation may explain the partial meiotic arrest of ROS-treated oocytes and the accelerated maturation observed after such treatment. Mol. Reprod. Dev. 69: 466,474, 2004. © 2004 Wiley-Liss, Inc. [source]

    Phosphoinositide 3-kinase mediated signalling contributes to development of diabetes-induced abnormal vascular reactivity of rat carotid artery

    Mariam H. M. Yousif
    Abstract Diabetes mellitus is associated with vascular complications, including an impairment of vascular function and alterations in the reactivity of blood vessels to vasoactive agents. Phosphatidylinositol 3-kinase (PI3K) is a signalling enzyme that plays key roles in vascular growth, proliferation and cellular apoptosis and is implicated in modulating vascular smooth muscle contractility. The aim of this study was to determine whether PI3K plays a role in development of diabetes-induced altered vascular reactivity to selected vasoconstrictors and vasodilators. The effect of 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), a selective PI3K inhibitor, on isolated segments of carotid arteries from streptozotocin (STZ)-diabetic rats was investigated. Ring segments of the isolated carotid arteries were mounted in organ baths to measure changes in isometric tension. Our results showed that STZ treatment produced an increase in the vasoconstrictor response to norepinephrine (NE), angiotensin II (Ang II) and endothelin-1 (ET-1) and an attenuated vasodilator response to carbachol and histamine in the isolated carotid arteries from STZ-diabetic animals. Diabetes-induced impaired vascular responsiveness to the vasoactive agonists was prevented by chronic inhibition of PI3K by LY294002 even though blood glucose levels remained high. This is the first study to show that selective inhibition of PI3K can attenuate the development of diabetes-induced abnormal vascular reactivity in the isolated carotid arteries of diabetic rats. Copyright © 2005 John Wiley & Sons, Ltd. [source]

    Post-ischaemic activation of kinases in the pre-conditioning-like cardioprotective effect of the platelet-activating factor

    ACTA PHYSIOLOGICA, Issue 3 2009
    C. Penna
    Abstract Aim:, Platelet-activating factor (PAF) triggers cardiac pre-conditioning against ischemia/reperfusion injury. The actual protection of ischaemic pre-conditioning occurs in the reperfusion phase. Therefore, we studied in this phase the kinases involved in PAF-induced pre-conditioning. Methods:, Langendorff-perfused rat hearts underwent 30 min of ischaemia and 2 h of reperfusion (group 1, control). Before ischaemia, group 2 hearts were perfused for 19 min with PAF (2 × 10,11 m); groups 3,5 hearts were co-infused during the initial 20 min of reperfusion, with the protein kinase C (PKC) inhibitor chelerythrine (5 × 10,6 m) or the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (5 × 10,5 m) and atractyloside (2 × 10,5 m), a mitochondrial permeability transition pore (mPTP) opener respectively. Phosphorylation of PKC,, PKB/A,t, GSK-3, and ERK1/2 at the beginning of reperfusion was also checked. Left ventricular pressure and infarct size were determined. Results:, PAF pre-treatment reduced infarct size (33 ± 4% vs. 64 ± 5% of the area at risk of control hearts) and improved pressure recovery. PAF pre-treatment enhanced the phosphorylation/activation of PKC,, PKB/A,t and the phosphorylation/inactivation of GSK-3, at reperfusion. Effects on ERK1/2 phosphorylation were not consistent. Infarct-sparing effect and post-ischaemic functional improvement induced by PAF pre-treatment were abolished by post-ischaemic infusion of either chelerythrine, LY294002 or atractyloside. Conclusions:, The cardioprotective effect exerted by PAF pre-treatment involves activation of PKC and PI3K in post-ischaemic phases and might be mediated by the prevention of mPTP opening in reperfusion via GSK-3, inactivation. [source]

    Migration of mesenchymal cell fated to blastema is necessary for fish fin regeneration

    Yuki Nakatani
    Urodeles and fish have higher regeneration ability in a variety of tissues and organs than do other vertebrate species including mammals. Though many studies have aimed at identifying the cellular and molecular basis for regeneration, relatively little is known about the detailed cellular behaviors and involved molecular basis. In the present study, a small molecule inhibitor was used to analyzed the role of phosphoinositide 3-kinase (PI3K) signaling during regeneration. We showed that the inhibitor disrupted the formation of blastema including the expression of characteristic genes. The failure of blastema formation was due to the impaired migration of mesenchymal cells to the distal prospective blastema region, although it had a little affect on cell cycle activation in mesenchymal cells. Moreover, we found that the epidermal remodeling including cell proliferation, distal cell migration and Akt phosphorylation was also affected by the inhibitor, implying a possible involvement of epidermis for proper formation of blastema. From these data, we propose a model in which distinct signals that direct the cell cycle activation, mesenchymal cell migration and epidermal remodeling coordinate together to accomplish the correct blastema formation and regeneration. [source]

    GDNF and insulin cooperate to enhance the proliferation and differentiation of enteric crest-derived cells

    Paul J. Focke
    Abstract Previously we have shown that glial derived neurotrophic factor (GDNF) stimulates modest increases in the proliferation of avian enteric crest-derived cells and similar increases in the phosphorylation of the phosphoinositide 3,kinase (PI3K) downstream substrate Akt (Akt-P). In the present study we tested whether GDNF-independent increases in PI3K activation would be sufficient to support proliferation. We found that insulin induces a large increase in the phosphorylation of Akt and can initiate DNA synthesis in avian enteric crest-derived cells, but is unable to maintain proliferation over time in culture, measured by BrdU incorporation. GDNF can also initiate DNA synthesis, but it too is unable to maintain BrdU incorporation in cultured enteric crest-derived cells. Sustained incorporation of BrdU after 16,48 h in culture is shown to be dependent on a combination of GDNF and insulin. Using a phospho-specific antibody, we found Akt-P levels to be similar in the proliferating (BrdU incorporation maintained from 16,48 h in culture) and nonproliferating populations, suggesting that Akt-P levels were not solely controlling the extent of BrdU incorporation. A minimum level of PI3K activation, however, is required, as shown by the dose-dependent reduction in proliferation with the PI3K inhibitor LY-294002. We conclude that the integrity of the PI3K pathway is essential for enteric crest-derived cell proliferation, but that the absolute levels of Akt-P do not determine the extent of proliferation. The enhanced proliferation in cultures containing both GDNF and insulin suggests that other pathways are involved, including the possibility that PI3K downstream effectors other than Akt are important in the regulation of avian enteric crest-derived cell proliferation. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 151,164, 2003 [source]

    Foxo1 regulates marginal zone B-cell development

    Jing Chen
    Abstract A fundamental component of signaling initiated by the BCR and CD19 is the activation of phosphoinositide 3-kinase. Downstream of phosphoinositide 3-kinase, the protein kinase AKT phosphorylates several substrates, including members of the forkhead box subgroup O (Foxo) transcription factor family. Among the Foxo proteins, Foxo1 has unique functions in bone marrow B-cell development and peripheral B-cell function. Here, we report a previously unrecognized role for Foxo1 in controlling the ratio of mature B-cell subsets in the spleen. Conditional deletion of Foxo1 in B cells resulted in an increased percentage of marginal zone B cells and a decrease in follicular (FO) B cells. In addition, Foxo1 deficiency corrected the absence of marginal zone B cells that occurs in CD19-deficient mice. These findings show that Foxo1 regulates the balance of mature B-cell subsets and is required for the marginal zone B-cell deficiency phenotype of mice lacking CD19. [source]

    Expression and function of NKG2D in CD4+ T cells specific for human cytomegalovirus

    Andrea Sáez-Borderías
    Abstract The human NKG2D killer lectin-like receptor (KLR) is coupled by the DAP10 adapter to phosphoinositide 3-kinase (PI3,K) and specifically interacts with different stress-inducible molecules (i.e. MICA, MICB, ULBP) displayed by some tumour and virus-infected cells. This KLR is commonly expressed by human NK cells as well as TCR,,+ and TCR,,+CD8+ T lymphocytes, but it has been also detected in CD4+ T cells from rheumatoid arthritis and cancer patients. In the present study, we analysed NKG2D expression in human cytomegalovirus (HCMV)-specific CD4+ T lymphocytes. In vitro stimulation of peripheral blood mononuclear cells (PBMC) from healthy seropositive individuals with HCMV promoted variable expansion of CD4+NKG2D+ T lymphocytes that coexpressed perforin. NKG2D was detected in CD28, and CD28dull subsets and was not systematically associated with the expression of other NK cell receptors (i.e. KIR, CD94/NKG2 and ILT2). Engagement of NKG2D with specific mAb synergized with TCR-dependent activation of CD4+ T cells, triggering proliferation and cytokine production (i.e. IFN-, and TNF-,). Altogether, the data support the notion that NKG2D functions as a prototypic costimulatory receptor in a subset of HCMV-specific CD4+ T lymphocytes and thus may have a role in the response against infected HLA class II+ cells displaying NKG2D ligands. [source]

    Airway inflammation: chemokine-induced neutrophilia and the class,I phosphoinositide 3-kinases

    Abstract Class,I phosphoinositide 3-kinases (PI3K) are known to play a significant role in neutrophil chemotaxis. However, the relative contributions of different PI3K isoforms, and how these impact on lung inflammation, have not been addressed. In vitro studies using wild-type and PI3K, knockout neutrophils demonstrated the major role of the ,,isoform in chemotactic but not chemokinetic events. This was confirmed by a model of direct chemokine instillation into the airways in vivo. Within all studies, a low yet significant degree of neutrophil movement in the absence of PI3K, could be observed. No role for the ,,isoform was demonstrated both in vitro and in vivo using PI3K, kinase-dead knock-in mice. Moreover, further studies using the broad-spectrum PI3K inhibitors wortmannin or LY294002 showed no other class,I PI3K isoforms to be involved in these chemotactic processes. Here, we identify a contributory PI3K-independent mechanism of neutrophil movement, yet demonstrate PI3K, as the pivotal mediator through which the majority of neutrophils migrate into the lung in response to chemokines. These data resolve the complexities of chemokine-induced neutrophilia and PI3K signaling and define the ,,isoform as a promising target for new therapeutics to treat airway inflammatory diseases. [source]

    Mechanism of insulin-like growth factor I-mediated proliferation of adult neural progenitor cells: role of Akt

    Haviryaji S. G. Kalluri
    Abstract Insulin-like growth factor I (IGF-I) is involved in the proliferation and differentiation of adult neural progenitor cells; however, the underlying mechanism is not clear. We analysed the involvement of the phosphatidylinositol 3-kinase/Akt and MEK/extracellular signal-regulated kinase (ERK) pathways in the IGF-I-mediated proliferation of rat neural progenitor cells. Stimulation of neural progenitor cells with IGF-I enhanced the phosphorylation of Akt but not ERK. Cell proliferation assay demonstrated that 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (phosphoinositide 3-kinase inhibitor) but not 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)-butadiene (U0126) (ERK inhibitor) inhibited the IGF-I-induced survival of cells, whereas fibroblast growth factor 2 (FGF-2) enhanced the IGF-I-mediated survival of cells. Consistent with the cell proliferation assay, 5,bromo-2-deoxy-uridine incorporation studies established a negative role for IGF-I in proliferation. However, FGF-2 (ERK activator) in the presence of IGF-I (Akt activator) increased the proliferation of cells. Accordingly, stimulation of the ERK pathway by FGF-2 induced the expression of cyclin D1, which is essential for the entry of cells into cell cycle, and IGF-I in the presence of FGF-2 up-regulated the expression of cyclin D1. IGF-I in the absence or presence of FGF-2 increased the phosphorylation of glycogen synthase kinase, thus supporting its role in the survival of neural progenitor cells. To further confirm the role of ERK activation in the proliferation, we cultured cells in FGF-2 + IGF-I-containing medium in the presence and absence of U0126 (ERK inhibitor), and showed the inhibition of nestin expression in U0126-treated cells. The decrease in the cyclin D1 content in conjunction with the inhibition of nestin expression by ERK inhibitor confirms the role of ERK in the proliferation of cells. [source]

    3T3-L1 adipocyte apoptosis induced by thiazolidinediones is peroxisome proliferator-activated receptor-,-dependent and mediated by the caspase-3-dependent apoptotic pathway

    FEBS JOURNAL, Issue 3 2010
    Yuanyuan Xiao
    Although thiazolidinediones (TZDs) are potent promoters of adipogenesis in the preadipocyte, they induce apoptosis in several other cell types, such as cancer cells, endothelial cells and T-lymphocytes. In this study, we investigated the proapoptotic effect of TZDs in mature 3T3-L1 adipocytes, which express high levels of the peroxisome proliferator-activated receptor-, (PPAR,) protein. Apoptosis was induced in mature 3T3-L1 adipocytes by treatment with troglitazone, pioglitazone or prostaglandin J2, and could be blocked by the PPAR, antagonist GW9662. Treatment with PPAR, agonists also decreased Akt-1 protein and phosphorylation levels without affecting phosphoinositide 3-kinase and PTEN. Further analysis indicated that in troglitazone-treated 3T3-L1 adipocytes, Bad phosphorylation and Bcl-2 protein levels were reduced, and Bax translocation to the mitochondria was increased. Subsequently, cytochrome c release and caspase-3 cleavage were observed. TZD-induced adipocyte apoptosis could be blocked by the caspase-3 inhibitor Ac-DEVD-CHO or by overexpression of Bcl2. In cultured rat primary adipocytes, similar apoptosis-inducing effects of troglitazone were also observed. Thus, TZDs promote apoptosis in adipocytes through a PPAR,-dependent pathway. This apoptosis is mediated by the inhibition of Akt-1, which decreases Bad phosphorylation and activates the mitochondrial apoptotic pathway. [source]

    Pfkfb3 is transcriptionally upregulated in diabetic mouse liver through proliferative signals

    FEBS JOURNAL, Issue 16 2009
    Joan Duran
    The ubiquitous isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (uPFK-2), a product of the Pfkfb3 gene, plays a crucial role in the control of glycolytic flux. In this study, we demonstrate that Pfkfb3 gene expression is increased in streptozotocin-induced diabetic mouse liver. The Pfkfb3/-3566 promoter construct linked to the luciferase reporter gene was delivered to the liver via hydrodynamic gene transfer. This promoter was upregulated in streptozotocin-induced diabetic mouse liver compared with transfected healthy cohorts. In addition, increases were observed in Pfkfb3 mRNA and uPFK-2 protein levels, and intrahepatic fructose-2,6-bisphosphate concentration. During streptozotocin-induced diabetes, phosphorylation of both p38 mitogen-activated protein kinase and Akt was detected, together with the overexpression of the proliferative markers cyclin D and E2F. These findings indicate that uPFK-2 induction is coupled to enhanced hepatocyte proliferation in streptozotocin-induced diabetic mouse liver. Expression decreased when hepatocytes were treated with either rapamycin or LY 294002. This shows that uPFK-2 regulation is phosphoinositide 3-kinase,Akt,mammalian target of rapamycin dependent. These results indicate that fructose-2,6-bisphosphate is essential to the maintenance of the glycolytic flux necessary for providing energy and biosynthetic precursors to dividing cells. [source]

    A novel N-terminal hydrophobic motif mediates constitutive degradation of serum- and glucocorticoid-induced kinase-1 by the ubiquitin,proteasome pathway

    FEBS JOURNAL, Issue 13 2006
    Agata M. Bogusz
    Serum- and glucocorticoid-induced protein kinase-1 (SGK-1) plays a critical role in regulation of the epithelial sodium channel, ENaC. SGK-1 also shares significant catalytic domain homology with protein kinase B (PKB/AKT-1) and is a downstream effector of antiapoptotic phosphoinositide 3-kinase signaling. Steady-state levels of an active SGK-1 are tightly regulated by rapid transcriptional activation and post-translational modification including phosphorylation. We show here that endogenous SGK-1 protein is polyubiquitinated and rapidly degraded by the 26S proteasome. In contrast to other rapidly degraded kinases, neither the catalytic activity of SGK-1 nor activation site phosphorylation was required for its ubiquitin modification and degradation. Instead, SGK-1 degradation required a lysine-less six-amino-acid (amino acids 19,24) hydrophobic motif (GMVAIL) within the N-terminal domain. Deletion of amino acids 19,24 significantly increased the half-life of SGK1 and prevented its ubiquitin modification. Interestingly, this minimal region was also required for the association of SGK-1 with the endoplasmic reticulum. Ubiquitin modification and degradation of SGK-1 were increasingly inhibited by the progressive mutation of six N-terminal lysine residues surrounding the GMVAIL motif. Mutation of all six lysines to arginine did not disrupt the subcellular localization of SGK-1 despite a significant decrease in ubiquitination, implying that this modification per se was not required for targeting to the endoplasmic reticulum. These results suggest that constitutive ubiquitin-mediated degradation of SGK-1 is an important mechanism regulating its biological activity. [source]

    Activation of p21-activated kinase 1 is required for lysophosphatidic acid-induced focal adhesion kinase phosphorylation and cell motility in human melanoma A2058 cells

    FEBS JOURNAL, Issue 8 2004
    In Duk Jung
    Lysophosphatidic acid (LPA), one of the naturally occurring phospholipids, stimulates cell motility through the activation of Rho family members, but the signaling mechanisms remain to be elucidated. In the present study, we investigated the roles of p21-activated kinase 1 (PAK1) on LPA-induced focal adhesion kinase (FAK) phosphorylation and cell motility. Treatment of human melanoma cells A2058 with LPA increased phosphorylation and activation of PAK1, which was blocked by treatment with pertussis toxin and by inhibition of phosphoinositide 3-kinase (PI3K) with an inhibitor LY294002 or by overexpression of catalytically inactive mutant of PI3K,, indicating that LPA-induced PAK1 activation was mediated via a Gi protein and the PI3K, signaling pathway. In addition, we demonstrated that Rac1/Cdc42 signals acted as upstream effector molecules of LPA-induced PAK activation. However, Rho-associated kinase, MAP kinase kinase 1/2 or phospholipase C might not be involved in LPA-induced PAK1 activation or cell motility stimulation. Furthermore, PAK1 was necessary for FAK phosphorylation by LPA, which might cause cell migration, as transfection of the kinase deficient mutant of PAK1 or PAK auto-inhibitory domain significantly abrogated LPA-induced FAK phosphorylation. Taken together, these findings strongly indicated that PAK1 activation was necessary for LPA-induced cell motility and FAK phosphorylation that might be mediated by sequential activation of Gi protein, PI3K, and Rac1/Cdc42. [source]

    Transport of phosphatidylinositol 3-phosphate into the vacuole via autophagic membranes in Saccharomyces cerevisiae

    GENES TO CELLS, Issue 6 2008
    Keisuke Obara
    Vps34, the sole PtdIns 3-kinase in yeast, is essential for autophagy. Here, we show that the lipid-kinase activity of Vps34 is required for autophagy, implying an essential role of its product PtdIns(3)P. The protein-kinase activity of Vps15, a regulatory subunit of the PtdIns 3-kinase complex, is also required for efficient autophagy. We monitored the distribution of PtdIns(3)P in living cells using a specific indicator, the 2xFYVE domain derived from mammalian Hrs. PtdIns(3)P was abundant at endosomes and on the vacuolar membrane during logarithmic growth phase. Under starvation conditions, we observed massive transport of PtdIns(3)P into the vacuole. This accumulation was dependent on the membrane dynamics of autophagy. Notably, PtdIns(3)P was highly enriched and delivered into the vacuole as a component of autophagosome membranes but not as a cargo enclosed within them, implying direct involvement of this phosphoinositide in autophagosome formation. We also found a possible enrichment of PtdIns(3)P on the inner autophagosomal membrane compared to the outer membrane. Based on these results we discuss the function of PtdIns(3)P in autophagy. [source]

    Tel2: a common partner of PIK-related kinases and a link between DNA checkpoint and nutritional response?

    GENES TO CELLS, Issue 12 2007
    Junko Kanoh
    A recent paper (Hayashi et al. 2007) in this issue of Genes to Cells shows that the fission yeast Schizosaccharomyces pombe Tel2, a homologue of mammalian/worm CLK2/Clk-2/Rad-5, physically interacts with all the phosphoinositide 3-kinase-related kinases (PIKKs) that include Rad3/Tel1 (ATR/ATM homologues), Tor1/Tor2 (TOR kinases) and Tra1/Tra2 (TRRAP homologues), raising the possibility that Tel2 family proteins link various PIKK-related cellular processes by interacting with PIKK family proteins. In this minireview, implications and impact of the findings, and a possibility that PIKKs are functionally related through Tel2, are discussed. [source]

    ,-catenin mediates insulin-like growth factor-I actions to promote cyclin D1 mRNA expression, cell proliferation and survival in oligodendroglial cultures

    GLIA, Issue 9 2010
    Ping Ye
    Abstract By promoting cell proliferation, survival and maturation insulin-like growth factor (IGF)-I is essential to the normal growth and development of the central nervous system. It is clear that IGF-I actions are primarily mediated by the type I IGF receptor (IGF1R), and that phosphoinositide 3 (PI3)-Akt kinases and MAP kinases signal many of IGF-I-IGF1R actions in neural cells, including oligodendrocyte lineage cells. The precise downstream targets of these signaling pathways, however, remain to be defined. We studied oligodendroglial cells to determine whether ,-catenin, a molecule that is a downstream target of glycogen synthase kinase-3, (GSK3,) and plays a key role in the Wnt canonical signaling pathway, mediates IGF-I actions. We found that IGF-I increases ,-catenin protein abundance within an hour after IGF-I-induced phosphorylation of Akt and GSK3,. Inhibiting the PI3-Akt pathway suppressed IGF-I-induced increases in ,-catenin and cyclin D1 mRNA, while suppression of GSK3, activity simulated IGF-I actions. Knocking-down ,-catenin mRNA by RNA interference suppressed IGF-I-stimulated increases in the abundance of cyclin D1 mRNA, cell proliferation, and cell survival. Our data suggest that ,-catenin is an important downstream molecule in the PI3-Akt-GSK3, pathway, and as such it mediates IGF-I upregulation of cyclin D1 mRNA and promotion of cell proliferation and survival in oligodendroglial cells. © 2010 Wiley-Liss, Inc. [source]

    PI3K-FRAP/mTOR pathway is critical for hepatocyte proliferation whereas MEK/ERK supports both proliferation and survival

    HEPATOLOGY, Issue 5 2002
    Alexandre Coutant
    Growth factors are known to favor both proliferation and survival of hepatocytes. In this work, we investigated the role of 2 main signaling pathways, phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MEK)/extracellular signal,regulated kinase (ERK), in these processes. First, evidence was provided that the PI3K cascade as well as the MEK/ERK cascade is a key transduction pathway controlling hepatocyte proliferation, as ascertained by arrest of DNA synthesis in the presence of LY294002, a specific PI3K inhibitor. Inhibition of FRAP/mTOR by rapamycin also abrogated DNA replication and protein synthesis induced by growth factor. We showed that expression of cyclin D1 at messenger RNA (mRNA) and protein levels was regulated by this pathway. We highlighted that 4E-BP1 phosphorylation was not activated by epidermal growth factor (EGF) but was under an insulin-regulation mechanism through a PI3K-FRAP/mTOR activation that could account for the permissive role of insulin on hepatocyte proliferation. No interference between the MEK/ERK pathway and 4E-BP1 phosphorylation was detected, whereas p70S6K phosphorylation induced by EGF was under a U0126-sensitive regulation. Last, we established that the antiapoptotic function of EGF was dependent on MEK, whereas LY294002 and rapamycin had no direct effect on cell survival. Taken together, these data highlight the regulation and the role of 2 pathways that mediate growth-related response by acting onto distinct steps. In conclusion, hepatocyte progression in late G1 phase induced by EGF generates survival signals depending on MEK activation, whereas PI3K and MEK/ERK cascades are both necessary for hepatocyte replication. [source]

    Characterization of the migration of lung and blood T cells in response CXCL12 in a three-dimensional matrix

    IMMUNOLOGY, Issue 4 2010
    Caroline E. Day
    Summary The ability of T cells to microlocalize within tissues, such as the lung, is crucial for immune surveillance and increased T-cell infiltration is a feature of many inflammatory lung conditions. T-cell migration has mainly been studied in two-dimensional assays. Using three-dimensional collagen gels to mimic the extracellular matrix of lung tissue, we have characterized the migration of T lymphocytes isolated from peripheral blood (PBT) and lung (LT) in response to interleukin-2 (IL-2) and CXCL12. Freshly isolated PBT and LT showed a low degree of migration (blood 4·0 ± 1·3% and lung 4·1 ± 1·7%). Twenty-four hours of culture increased the percentage of migrating PBT and LT (blood 17·5 ± 2·9% and lung 17·7 ± 3·8%). The IL-2 stimulation modestly increased migration of PBT after 6 days (32·3 ± 6·0%), but had no effect on the migration of LT (25·5 ± 3·2%). Twenty-four hours of stimulation with anti-CD3/CD28 caused a small but significant increase in the migration of PBT (to 36·4 ± 5·8%). In a directional three-dimensional assay, CXCL12 failed to induce migration of fresh PBT or LT. Twenty-four hours of culture, which increased CXCR4 expression of PBT 3·6-fold, significantly increased the migration of PBT in response to CXCL12. Migration of PBT to CXCL12 was blocked by pertussis toxin, but not by the phosphoinositide 3-kinase inhibitor wortmannin. Twenty-four-hour cultured LT did not respond to CXCL12. CD3/CD28-stimulation inhibited CXCL12-mediated migration of PBT. These results suggest that the migration pattern of PBT is distinct from that of LT. [source]

    Protein kinase B modulates the sensitivity of human neuroblastoma cells to insulin-like growth factor receptor inhibition

    Ana S. Guerreiro
    Abstract The potential of the novel insulin-like growth factor receptor (IGF-IR) inhibitor NVP-AEW541 as an antiproliferative agent in human neuroblastoma was investigated. Proliferation of a panel of neuroblastoma cell lines was inhibited by NVP-AEW541 with IC50 values ranging from 0.15 to 5 ,M. Experiments using an IGF-IR neutralizing antibody confirmed that the IGF-IR was essential to support growth of neuroblastoma cell lines. The expression levels of the IGF-IR in individual neuroblastoma cell lines did not correlate with the sensitivities to NVP-AEW541, while coexpression of the IGF-IR and the insulin receptor (IR) correlated with lower sensitivity to the inhibitor in some cell lines. Intriguingly, high levels of activation of Akt/protein kinase B (PKB) and phosphorylation of the ribosomal S6 protein were observed in neuroblastoma cell lines with decreased sensitivities to NVP-AEW541. Inhibition of Akt/PKB activity restored the sensitivity of neuroblastoma cells to the IGF-IR inhibitor. Transfection of neuroblastoma cells with activated Akt or ribosomal protein S6 kinase (S6K) decreased the sensitivity of the cells to NVP-AEW541. IGF-I-stimulated proliferation of neuroblastoma cell lines was completely blocked by NVP-AEW541, or by a combination of an inhibitor of phosphoinositide 3-kinase and rapamycin. In addition to its antiproliferative effects, NVP-AEW541 sensitized neuroblastoma cells to cisplatin-induced apoptosis. Together, our data demonstrate that NVP-AEW541 in combination with Akt/PKB inhibitors or chemotherapeutic agents may represent a novel approach to target human neuroblastoma cell proliferation. © 2006 Wiley-Liss, Inc. [source]

    Regulation of phosphoinositide signaling by the inositol polyphosphate 5-phosphatases

    IUBMB LIFE, Issue 8 2006
    Megan V. Astle
    Abstract Phosphoinositide signaling molecules control cellular growth, proliferation and differentiation, intracellular vesicle trafficking, and cytoskeletal rearrangement. The inositol polyphosphate 5-phosphatase family remove the D-5 position phosphate from PtdIns(3,4,5)P3, PtdIns(4,5)P2 and PtdIns(3,5)P2 forming PtdIns(3,4)P2, PtdIns(4)P and PtdIns(3)P respectively. This enzyme family, comprising ten mammalian members, exhibit seemingly non-redundant functions including the regulation of synaptic vesicle recycling, hematopoietic cell function and insulin signaling. Here we highlight recently established insights into the functions of two well characterized 5-phosphatases OCRL and SHIP2, which have been the subject of extensive functional studies, and the characterization of recently identified members, SKIP and PIPP, in order to highlight the diverse and complex functions of this enzyme family. iubmb Life, 58: 451 - 456, 2006 [source]

    Phosphoinositide 3-kinase is not overexpressed in melanocytic lesions

    Rajendra S. Singh
    Background:, Although various studies have stressed the role of phosphatase and tensin homologue deleted on chromosome 10 (PTEN)-PI3K-AKT pathway in the progression of melanocytic lesions, little is known about the expression pattern of PI3K in these lesions. Objective:, To investigate the expression pattern of PI3K in benign and dysplastic nevi, primary melanomas, and metastatic melanomas and the role of PTEN and PI3K in melanocytic tumor progression. Methods:, Tissue microarrays were constructed using formalin-fixed, paraffin-embedded archival tissue blocks from 89 melanocytic lesions: 17 benign nevi, 18 dysplastic nevi, 23 primary melanomas, and 31 metastatic melanomas. Expression of PTEN and PI3K (p85 and p110 subunits) was evaluated immunohistochemically, and the number of cells and labeling intensity were assessed semiquantitatively. Results:, Both benign and dysplastic nevi showed strong cytoplasmic staining with PTEN, which was subsequently less in melanomas and completely lost in the metastatic lesions. Eleven of 17 (64%) benign nevi, seven of 10 (70%) dysplastic nevi, four of 23 (17%) primaries, and one of 31 (3%) visceral or lymph node metastasis showed strong positivity. Loss of PTEN expression from benign and dysplastic nevi to melanoma was statistically significant (p = 0.001). Although few cells showed reactivity for phosphoinositide 3-kinase (PI3 kinase)-p85 subunit, strong positivity was not detected in the cytoplasm of benign, malignant, or metastatic lesions, except for a single visceral metastasis. Three of 13 (23%) nevi showed positivity for the p110 subunit. No positivity was observed in the dysplastic nevi. Two of 22 (9%) melanomas, one of 14 (7%) visceral metastasis, and three of 12 (25%) lymph node metastasis showed strong positivity. There was no statistical difference in PI3 kinase expression in benign and malignant melanocytic lesions (p = 0.2). Conclusion:, PI3K is not overexpressed in melanocytic lesions. [source]

    KATP channel blockade protects midbrain dopamine neurons by repressing a glia-to-neuron signaling cascade that ultimately disrupts mitochondrial calcium homeostasis

    Damien Toulorge
    J. Neurochem. (2010) 114, 583,564. Abstract While KATP channels serve primarily as metabolic gatekeepers in excitable cells, they might also participate in other important cellular functions. Here, we demonstrate that KATP channel blockade with the sulfonylurea derivative glibenclamide provided robust protection to dopamine neurons undergoing spontaneous and selective degeneration in midbrain cultures. Unexpectedly, glibenclamide operated not by a direct effect on dopamine neurons but instead by halting the proliferation of a population of immature glial cells lacking astrocytic and microglial markers. The antimitotic effect of glibenclamide appeared essential to unmask a prosurvival phosphoinositide 3-kinase (PI3K)/Akt-dependent signaling pathway that controlled shuttling of calcium from endoplasmic reticulum to mitochondria in dopamine neurons. Preventing integrin-ligand interactions with a decoy ligand, the Arg-Gly-Asp-Ser sequence peptide, reproduced survival promotion by glibenclamide via a mechanism that also required PI3K/Akt-dependent regulation of mitochondrial calcium. Noticeably, Arg-Gly-Asp-Ser did not cause a reduction in glial cell numbers indicating that it prevented the death process downstream of the level at which glibenclamide intervenes. Based on these results, we propose that KATP channel blockade protected dopamine neurons by inhibiting a glia-to-neuron signaling pathway that propagates through integrin/ligand interactions and ultimately disrupts PI3K/Akt-dependent signaling and mitochondrial calcium homeostasis. [source]

    Bi-directional modulation of fast inhibitory synaptic transmission by leptin

    Natasha Solovyova
    Abstract The hormone leptin has widespread actions in the CNS. Indeed, leptin markedly influences hippocampal excitatory synaptic transmission and synaptic plasticity. However, the effects of leptin on fast inhibitory synaptic transmission in the hippocampus have not been evaluated. Here, we show that leptin modulates GABAA receptor-mediated synaptic transmission onto hippocampal CA1 pyramidal cells. Leptin promotes a rapid and reversible increase in the amplitude of evoked GABAA receptor-mediated inhibitory synaptic currents (IPSCs); an effect that was paralleled by increases in the frequency and amplitude of miniature IPSCs, but with no change in paired pulse ratio or coefficient of variation, suggesting a post-synaptic expression mechanism. Following washout of leptin, a persistent depression (inhibitory long-lasting depression) of evoked IPSCs was observed. Whole-cell dialysis or bath application of inhibitors of phosphoinositide 3 (PI 3)-kinase or Akt prevented leptin-induced enhancement of IPSCs indicating involvement of a post-synaptic PI 3-kinase/Akt-dependent pathway. In contrast, blockade of PI 3-kinase or Akt activity failed to alter the ability of leptin to induce inhibitory long-lasting depression, suggesting that this process is independent of PI 3-kinase/Akt. In conclusion these data indicate that the hormone leptin bi-directionally modulates GABAA receptor-mediated synaptic transmission in the hippocampus. These findings have important implications for the role of this hormone in regulating hippocampal pyramidal neuron excitability. [source]

    Noradrenaline enhances the expression of the neuronal monocarboxylate transporter MCT2 by translational activation via stimulation of PI3K/Akt and the mTOR/S6K pathway

    Julie Chenal
    Abstract Monocarboxylate transporter 2 (MCT2) expression is up-regulated by noradrenaline (NA) in cultured cortical neurons via a putative but undetermined translational mechanism. Western blot analysis showed that p44/p42 mitogen-activated protein kinase (MAPK) was rapidly and strongly phosphorylated by NA treatment. NA also rapidly induced serine/threonine protein kinase from AKT virus (Akt) phosphorylation but to a lesser extent than p44/p42 MAPK. However, Akt activation persisted over a longer period. Similarly, NA induced a rapid and persistent phosphorylation of mammalian target of rapamycin (mTOR), a kinase implicated in the regulation of translation in the central nervous system. Consistent with activation of the mTOR/S6 kinase pathway, phosphorylation of the ribosomal S6 protein, a component of the translation machinery, could be observed upon treatment with NA. In parallel, it was found that the NA-induced increase in MCT2 protein was almost completely blocked by LY294002 (phosphoinositide 3-kinase inhibitor) as well as by rapamycin (mTOR inhibitor), while mitogen-activated protein kinase kinase and p38 MAPK inhibitors had much smaller effects. Taken together, these data reveal that NA induces an increase in neuronal MCT2 protein expression by a mechanism involving stimulation of phosphoinositide 3-kinase/Akt and translational activation via the mTOR/S6 kinase pathway. Moreover, considering the role of NA in synaptic plasticity, alterations in MCT2 expression as described in this study might represent an adaptation to face energy demands associated with enhanced synaptic transmission. [source]

    Regulation of mitogen-activated protein kinases by glutamate receptors

    John Q. Wang
    Abstract Glutamate receptors regulate gene expression in neurons by activating intracellular signaling cascades that phosphorylate transcription factors within the nucleus. The mitogen-activated protein kinase (MAPK) cascade is one of the best characterized cascades in this regulatory process. The Ca2+ -permeable ionotropic glutamate receptor, mainly the NMDA receptor subtype, activates MAPKs through a biochemical route involving the Ca2+ -sensitive Ras-guanine nucleotide releasing factor, Ca2+/calmodulin-dependent protein kinase II, and phosphoinositide 3-kinase. The metabotropic glutamate receptor (mGluR), however, activates MAPKs primarily through a Ca2+ -insensitve pathway involving the transactivation of receptor tyrosine kinases. The adaptor protein Homer also plays a role in this process. As an information superhighway between surface glutamate receptors and transcription factors in the nucleus, active MAPKs phosphorylate specific transcription factors (Elk-1 and CREB), and thereby regulate distinct programs of gene expression. The regulated gene expression contributes to the development of multiple forms of synaptic plasticity related to long-lasting changes in memory function and addictive properties of drugs of abuse. This review, by focusing on new data from recent years, discusses the signaling mechanisms by which different types of glutamate receptors activate MAPKs, features of each MAPK cascade in regulating gene expression, and the importance of glutamate/MAPK-dependent synaptic plasticity in memory and addiction. [source]

    Activation of phosphoinositide-3 kinase/Akt pathway by FeSO4 in rat cerebral cortex synaptic endings

    Romina M. Uranga
    Abstract The aim of this work was to study the involvement of the phosphoinositide-3-kinase (PI3K)/Akt pathway in synaptic endings incubated under oxidative stress conditions. Synaptosomes purified from rat cerebral cortex were exposed to FeSO4 (50 ,M) for different periods of time. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction and lactate dehydrogenate (LDH) leakage were significantly affected after 5 min of incubation in the presence of FeSO4, with respect to control conditions. In whole synaptosomes incubated in the presence of [,- 32P]ATP, phosphoinositide (PPI) labeling was increased after 5 min of Fe2+ exposure. This effect was prevented by the specific PI3K inhibitor LY294002. Anti-p85 immunoprecipitates (IPs) obtained from synaptosomes preincubated with Fe2+ (5 min) showed a PI3K activity two-fold higher than the activity recovered under control conditions. Additionally, Akt activation was temporally coincident with PI3K activation. LY294002 was not able to prevent the LDH leakage and diminution of MTT reduction induced by Fe2+. Our results demonstrate that free iron provokes the early activation of PI3K/Akt pathway, but this activation is not sufficient for protecting synaptic endings from oxidative damage. © 2007 Wiley-Liss, Inc. [source]